Near-infrared cyanine dyes and conjugates thereof

Abstract

To provide compounds of the cyanine family having near-infrared emission exhibiting improved physicochemical and biological properties, and to provide conjugates thereof with biological ligands.SOLUTION: Cyanine derivatives of Formula (I) are provided. It may be suitably conjugated to an appropriate targeting moiety via an appropriate functional group which acts as a site of attachment.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of optical imaging. More specifically, the present invention relates to compounds of the cyanine family with near-infrared emission and their conjugates with biological ligands, characterized by improved physicochemical and biological properties. The present invention also relates to the use of these compounds as optical diagnostic agents in the imaging or therapy of solid tumors, their preparation methods, and compositions containing them. [Background technology]

[0002] Dyes are chemical compounds that absorb photons of a specific wavelength upon photoexcitation and re-emit a portion of that energy, usually at a longer wavelength, depending on their quantum efficiency. Specifically, cyanine dyes are fluorescent organic molecules characterized by a delocalized electron system confined between two nitrogen atoms and spanning a polymethine bridge. Some of them have favorable optical properties, low toxicity, and good solubility in aqueous media, making them useful as contrast agents for biomedical imaging. Cyanine dyes that emit in the near-infrared region (700-900 nm) are particularly useful for biomedical imaging applications due to their higher penetration depth compared to dyes that fluoresce in the visible spectrum.

[0003] Among near-infrared dyes used in biomedical imaging, indocyanine green (ICG) is currently the only drug approved for human use. Due to its strong binding to plasma proteins (blood pool effect) and rapid clearance of the unbound fraction by the liver (Cherrick et al., J Clin Invest 1960;39(4):592-600), ICG is routinely used for assessing tissue perfusion and angiographic applications. Furthermore, ICG is also being tested as an investigational drug for tumor imaging during diagnostic and interventional (fluorescence-guided surgical) procedures. ICG distributes and accumulates in tumor tissue by a combination of passive diffusion and the enhanced permeability and retention (EPR) effect (Onda N. et al., Int J Cancer 2016;139,673-682). False positives are a common clinical finding associated with the use of ICG for tumor imaging due to its nonspecific accumulation properties (Tummers Q. et al., PlosOne 2015;10(6):e0129766).

[0004] To improve the sensitivity and specificity of detection, additional contrast agents for near-infrared imaging are being developed that utilize dyes conjugated to carrier moieties (i.e., biomolecules) that target overexpressed tumor epitopes (Achilefu S. et al., J Med Chem 2002;45,2003-2015). For example, ICG and S0456 are examples of near-infrared dyes conjugated to tumor-targeting moieties and are currently being tested in clinical trials for intraoperative tumor detection (Fidel J. et al., Cancer Res. 2015;15;75(20):4283-4291; Hogstins C. et al., Clin Cancer Res 2016;22(12);2929-38).

[0005] Despite various attempts to find suitable imaging agents, there remains a need for improved dyes that offer optimal solubility and low aggregation in aqueous media, high fluorescence efficiency, and optimal biological properties. The biological properties of dyes, particularly their binding affinity to plasma proteins such as albumin, can strongly influence their distribution and tissue accumulation upon in vivo administration. For example, dyes with high binding affinity to human albumin are sequestered in the plasma compartment after intravenous administration and have low tissue extravasation rates, severely limiting their diagnostic applications. Furthermore, the biological properties of dyes can affect the tissue distribution of conjugates consisting of the dye itself and a biomolecule targeting a biological epitope on pathological tissue. Near-infrared dyes with low binding affinity to human serum albumin and nonspecific accumulation are preferred for in vivo applications. This need is most critical when the dye is conjugated to a biomolecule that specifically binds to a molecular epitope or pathological tissue (e.g., tumor). The present invention addresses these and other needs.

[0006] WO2002 / 024815 and WO2007 / 136996 (Li-Cor Inc.) and WO2004 / 065491 (Schering AG) report stable cyanine dyes useful for optical imaging applications, featuring high solubility in aqueous media and functional groups for direct conjugation with biomolecules. However, no teachings are provided on how to obtain dyes with optimal biological properties.

[0007] WO2015 / 114171 discloses small molecule targeted drug conjugates for the delivery of drugs that inhibit cancer cells. In particular, it reports the IRDye 750 conjugate "C6" for use in flow cytometry analysis and in vivo imaging of tumors.

[0008] Wada H. et al., Chemical Engineering Journal 2018, 340(3):51-57, discloses a NIR fluorescent nanoprobe using a mannose-conjugated ZW800-1 derivative for intraoperative pan-lymph node mapping and real-time optical imaging.

[0009] Vendrell M. et al., Organic & Biomolecular Chemistry 2011, 9(13):4760-4762, reported that the NIR fluorescent deoxyglucose analog CyNE2-DG showed preferential uptake in cancer cells and was validated as an optical agent for tumor imaging. Summary of the Invention [Problem to be solved by the invention]

[0010] Despite various attempts to find suitable imaging agents, there remains a need for improved dyes that offer optimal stability and fluorescence efficiency, as well as optimal physicochemical and biological properties, and are designed for optical imaging of living organisms. This need is particularly critical when the dyes are conjugated to biomolecules that specifically bind to molecular epitopes or pathological tissues (e.g., tumors). The present invention addresses these and other needs. [Means for solving the problem]

[0011] Generally, the present invention aims to provide novel cyanine dyes, or their corresponding conjugates to binding moieties, that are useful as contrast medium for optical imaging and that aim to solve the above-mentioned problems.

[0012] The novel cyanine derivatives described herein surprisingly have outstanding optical properties and high solubility in aqueous media.Surprisingly, the compounds of the present invention have been found to have a very low binding affinity for human albumin compared to near-infrared dyes known in the prior art, which is particularly advantageous when these compounds are used after intravenous administration; said low affinity prevents the sequestration of the compounds in the plasma compartment by large proteins present in the blood, such as albumin, and the resulting reduction in the fraction of free dye available for efficient extravasation and distribution in the extracellular space.

[0013] The novel cyanine dyes can be suitably conjugated to suitable targeting moieties via suitable functional groups that act as binding sites, thus providing highly specific and sensitive contrast agents for molecular imaging. The low albumin binding affinity of the compounds of the present invention is particularly important in the case of dye-conjugates, because only their free fraction (not bound to albumin) can effectively interact with molecular targets.

[0014] A further aspect of the invention relates to such dyes as diagnostic agents for use in methods of optical imaging, in particular for use in optical imaging of human or animal organs or tissues, wherein the imaging is tomographic imaging of the organ and the monitoring of organ function includes methods of angiography, tissue perfusion imaging, urinary tract imaging, biliary imaging, neuroimaging, intraoperative cancer identification, fluorescence-guided surgery, fluorescence endoscopy, fluorescence laparoscopy, robotic surgery, open field surgery, laser-guided surgery, photodynamic therapy, fluorescence lifetime imaging, or photoacoustic or sonofluorescence.

[0015] The present invention further relates to manufacturing processes for the preparation of the provided dyes, corresponding conjugates and / or pharmaceutically acceptable salts thereof, as well as their use in the preparation of diagnostic agents.

[0016] According to a further aspect, the present invention relates to a pharmaceutically acceptable composition comprising at least one dye or dye-conjugate compound of the present invention, or a pharmaceutically acceptable salt thereof, in admixture with one or more physiologically acceptable carriers or excipients, said composition being particularly useful as an optical imaging agent for providing useful imaging of human or animal organs or tissues.

[0017] In another aspect, the present invention relates to a method for optical imaging of an organ, tissue or region of the body by use of optical imaging techniques comprising the use of an effective dose of a compound of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] Therefore, a first object of the present invention is to provide a compound of formula (I): JPEG2026012682000001.jpg72166 During the ceremony, X is a direct bond or -O-; Y is a group selected from linear or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclyl substituted by at least two hydroxyl groups; R1 and R2 are each independently a straight or branched C1-C6 alkyl substituted with a group selected from -SO3H, -COOH, -CONH2, and -COO-C1-C6 alkyl; and R3 is hydrogen, -SO3H, or a linear or branched C1-C6 alkyl substituted by -COOH or -CONH-Y, wherein Y is a group selected from linear or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclyl substituted by at least two hydroxyl groups; or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0019] Another object of the present invention is to provide the corresponding conjugate dyes represented by compounds of formula (II): JPEG2026012682000002.jpg76166 During the ceremony, X is a direct bond or -O-; Y is a group selected from linear or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclyl substituted by at least two hydroxyl groups; R1 is a linear or branched C1-C6 alkyl substituted by a group selected from -SO3H, -COOH, -CONH2 and -COO-C1-C6 alkyl; R4 is -SO3H, -COOH and -CONH-(S) m -T, wherein: S is a spacer; T is a targeting moiety; m is an integer equal to 0 or 1; and R5 is a straight or branched C1-C6 alkyl substituted with hydrogen, -SO3H, -COOH or -CONH-Y, and the group CONH-(S) m -T (R5 is selected from hydrogen, -SO3H, a linear or branched C1-C6 alkyl, substituted by -COOH or -CONH-Y, and a group CONH-(S) m -T), wherein Y, S, T and m are as defined above; wherein at least one of R4 and R5 is CONH-(S) m - a straight or branched C1-C6 alkyl substituted by T; or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0020] The present invention also relates to methods for preparing compounds of formula (I) or (II) using synthetic transformation steps.

[0021] The present invention also includes compounds of formula (I) or (II) for use as fluorescent probes for biomedical optical imaging applications.

[0022] definition For the purposes of this description, unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings:

[0023] The expression "straight-chain or branched C1-C6 alkyl" refers to an aliphatic hydrocarbon radical group having from 1 to 6 carbon atoms in the chain, which may be straight-chained or branched. For example, "C4 alkyl" includes within its meaning straight-chained or branched chains containing 4 carbon atoms. Similarly, "C1-C 20 "Alkyl" is an alkyl containing 1 to 20 carbon atoms. Representative preferred alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, pentyl, and hexyl. Unless otherwise specified, a straight or branched C1-C6 alkyl is a monovalent radical group. In some cases, it may be a "divalent" or "polyvalent" radical group in which two or more hydrogen atoms have been removed from the hydrocarbon radical group (e.g., methylene, ethylene, iso-propylene, etc.).

[0024] As used herein, the term "C3-C7 cycloalkyl" includes within its meaning saturated (i.e., alicyclic) carbocyclic rings containing from 3 to 7 carbon atoms. Suitable examples include C5-C7 carbocyclic rings, such as cyclohexyl rings.

[0025] The term "heterocyclyl" as used herein includes saturated alicyclic rings, preferably 5- to 7-membered saturated rings, further containing heteroatoms in the cyclic chain selected from N, O and S. Preferably, it refers to tetrahydropyran.

[0026] The term "hydroxyalkyl" refers to any corresponding alkyl chain in which one or more hydrogen atoms have been replaced by a hydroxyl group.

[0027] The term "alkoxy" includes within its meaning alkyl chains as defined above which further contain one or more oxygen atoms; examples include alkyl-oxy groups such as, for example, methoxy, ethoxy, n-propoxy, iso-propoxy, and alkyl-(poly)oxy groups in which the alkyl chain is interrupted by one or more oxygen atoms.

[0028] In the present description, the term "protecting group" (Pg) refers to a protecting group adapted to preserve the function of the group to which it is attached. In particular, protecting groups are used to preserve amino, hydroxyl or carboxyl functions. Suitable protecting groups include, for example, benzyl, carbonyl, such as formyl, 9-fluoromethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), t-butoxycarbonyl (Boc), isopropyloxycarbonyl or allyloxycarbonyl (Alloc), alkyl, such as tert-butyl or triphenylmethyl, sulfonyl, acetyl groups, such as trifluoroacetyl, benzyl esters, allyl, or other substituents commonly used for the protection of such functions and known to those skilled in the art (see, for example, the general references T.W. Green and P.G. Mughuts, Protective Groups in Organic Synthesis, Wiley, NY 2007, 4 th (See Ed., Ch.5).

[0029] Furthermore, the present invention also includes precursor or intermediate compounds suitable for preparing the desired compound of formula (I) or its salt. In such derivatives, the functional groups of R1-R5, such as carboxylic acid or carboxamide, can be protected with a suitable protecting group (Pg) as defined above, preferably an alkyl or ester group. If necessary, the hydroxyl group of the Y group can also be protected with a suitable protecting group (Pg) during the preparation of the compound of formula (I) or (II), thus forming, for example, an acetoxy, alkoxy, or ester group.

[0030] The expression "coupling reagent" refers to a reagent used, for example, in the formation of an amide bond between a carboxyl moiety and an amino moiety. The reaction can consist of two successive steps: activation of the carboxyl moiety followed by acylation of the amino group with an activated carboxylic acid.Non-limiting examples of such coupling agents include: carbodiimides, such as N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC); phosphonium reagents, such as (benzotriazol-1-yloxy)tris(dimethylaminopropyl); (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate (PyAOP), [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), and 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT); and aminium / uronium-imonium reagents such as N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU), N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (ΗATU), O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), 1-[1-(cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate (COMU) and fluoro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (TFFH) or other compounds known to those skilled in the art.

[0031] The term "small molecule" is used broadly herein to refer to an organic, inorganic, or organometallic compound having a molecular weight of less than about 5,000 daltons and capable of modulating a biological process or producing a biological effect when administered to an animal, including a human. The term "small molecule" can also be used interchangeably with the term "drug" or "biologically active moiety." For example, it can include any drug, active molecule, or compound that provides a beneficial effect on tumors and produces a local or systemic effect in a patient by binding to a specific biological target. Small molecule can also refer to a portion or residue of a parent drug that has been activated or chemically modified and covalently attached to a conjugated dye of the present invention.

[0032] The phrase "activated carboxylic acid" refers to a derivative of a carboxyl group that is more susceptible to nucleophilic attack than the free carboxyl group; suitable derivatives can include, for example, acid anhydrides, thioesters, acyl halides, NHS esters, and sulfo-NHS esters.

[0033] Furthermore, the term "moiety" or "residue" is intended herein to define the remainder of a given molecule when suitably attached or conjugated to the remainder of the molecule directly or via a suitable linker and / or spacer.

[0034] Targeting part (T) According to the present invention, a targeting moiety (T) is a molecule that binds to a biological target with particular selectivity and promotes the accumulation of an imaging agent in a specific tissue or body site. Generally, it is represented by a natural or synthetic molecule for use in biological systems.

[0035] Such specific binding can be achieved via a ligand, such as, for example, a small molecule, a protein, a peptide, a peptidomimetic, an enzyme substrate, an antibody or fragment thereof, or an aptamer, which interacts with a specific biological target expressed on the surface of the tissue or cell of interest.

[0036] Suitable biological targets for the compounds of the invention include, for example, epidermal growth factor (EGF) receptors such as EGFR or HER2; vascular endothelial growth factor (VEGF) receptors such as VEGFR1 or VEGFR2; carbonic anhydrase (CA) enzymes such as CAIX, CAII, or CAXII; mucin glycoproteins such as MUC1; glucose transporters such as GLUT-1; sodium-hydrogen exchange transporters such as NHE1; oncofetal glycoproteins such as carcinoembryonic antigen (CEA); chemokine receptors such as chemokine receptor type 4 (CXCR4); receptors for transferrin; ephrin receptors such as EPHA2; receptors for folate such as FR-α; glycoproteins that bind hyaluronan such as CD44; bombesin receptors such as BB1, BB2, and BB3; N-acetyl-L-aspartyl-L-glutamate (NAAG) peptidases such as prostate-specific membrane antigen (PSMA); and, especially, α v β3, α v β5, α v It may be an integrin receptor, such as a β6 or α5β1 integrin receptor.

[0037] For example, integrin receptor targeting moieties are represented by linear or cyclic peptides containing the sequence Arg-Gly-Asp (RGD). This tripeptide has high receptor binding specificity and is recognized as a ligand by the integrin receptor family present in the cell membrane. In fact, this RGD motif has been identified in several extracellular matrix glycoproteins, such as fibronectin or vitronectin, which mediate cell adhesion.

[0038] Thus, linear and cyclic peptides and peptidomimetics containing the sequence Arg-Gly-Asp (RGD), such as cRGD, cRGDfK, cRGDyK, cRGDfC, RGD-4C, RGD-2C, AH111585, NC100692, RGD-K5 (Kapp et al., Sci Rep, 2017, 7:3905), or their analogs and derivatives, are known examples of binding motifs that target cancer tissues in which cell membrane integrins are upregulated compared to healthy tissues.

[0039] In one embodiment, the compounds of the present invention can be conjugated to vectors known to target prostate-specific membrane antigen (PSMA), thus enabling the detection and imaging of prostate cancer.These ligands are represented, for example, by the vector glutamic acid-urea-lysine (EuK) or other PSMA binding vectors of the formula "EuX" described in EP3636635A1, that is, glutamic acid is linked to another amino acid or analog via a bridging urea, such as EuFA (glutamic acid-urea-3-(2-furyl)-alanine), EuPG (glutamic acid-urea-2-(2'-propynyl)-alanine), EuE (glutamic acid-urea-glutamic acid), or other urea-based peptidomimetics, such as EuK-(3-(2-naphthyl)-alanine)-tranexamic acid described in Benesova et al., J Nucl Med 2015,56:914-920.

[0040] In another embodiment, the compounds of the present invention can be conjugated to other small molecules, peptides, proteins, or antibodies, such as monoclonal antibodies, that are already used in therapy.Small molecules, including the drug acetazolamide, such as compounds 4a, 5a, 6a, 7a, and 8a (Wichert et al., Nat Chem 2015, 7:241-249), or their analogs and derivatives, are examples of small molecules that target the enzyme CAIX.Linear and cyclic peptides and peptidomimetics, such as peptide GE11 (described in Li et al., FASEB J 2005, 19:1978-85) and / or peptide L1 (described in Williams et al., Chem Biol Drug Des 2018, 91:605-619), or their analogs and derivatives, are examples of peptides that target epidermal growth factor receptor (EGFR). Among proteins, derivatives of epidermal growth factor (EGF) are examples of small proteins that target the epidermal growth factor receptor (EGFR). Among antibodies, panitumumab and cetuximab are examples of monoclonal antibodies that target the epidermal growth factor receptor (EGFR).

[0041] Preferably, the targeting ligand of the present invention can selectively bind to tumor cells or tissues.In particular, they can bind to tumors selected from brain tumors, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, esophageal cancer, skin cancer, stomach cancer, pancreatic cancer, bladder cancer, oral cancer, lung cancer, kidney cancer, uterine cancer, thyroid cancer, liver cancer, and colorectal cancer.In addition, the targeting ligand can bind to the metastatic spread of the above-mentioned cancers in tissues and organs different from the original origin.In addition, the targeting ligand can bind to preneoplastic lesions and dysplasias in different tissues and organs.

[0042] Spacer S According to the present invention, S is a spacer (may or may not exist) that separates the targeting moiety from the dye.The presence of a spacer is particularly relevant for some embodiments where there is a risk that the targeting moiety and the dye will negatively interact with each other.In addition, the presence of a spacer may be necessary when the dye is relatively large and may hinder the binding of the targeting moiety to the target site.

[0043] The spacer can be either flexible (e.g., a simple alkyl chain) or rigid (e.g., a cycloalkyl or aryl chain) to orient the dye away from the target. The spacer can also modify the pharmacokinetics and metabolism of the conjugate of formula (I) used as an imaging agent in vivo.

[0044] A hydrophilic spacer can reduce interactions with plasma proteins, shorten blood circulation time, and promote excretion. For example, when the spacer is a polyethylene glycol (PEG) moiety, the pharmacokinetics and blood clearance rate of the imaging agent in vivo can be modified. In such embodiments, the spacer can improve the clearance of the imaging agent from background tissues (i.e., muscle, blood), thus providing better diagnostic images due to high target-to-background contrast. Furthermore, the introduction of certain hydrophilic spacers can shift the excretion of the contrast agent from the liver to the kidney, thus reducing its overall retention in the body.

[0045] Thus, in one preferred embodiment, the spacer is C1-C 20 A hydrophilic moiety containing an alkyl, C3-C7 cycloalkyl, or aryl group. Preferably, the spacer is -(CH2) p COO-, -(CH2CH2O) p CH2CH2COO- and -(CH2CH2O) p CH2CH2NH-, where p is an integer between 0 and 20. Preferably, p is 2, 6, or 12.

[0046] If not required, the spacer is preferably absent, ie m is 0 and S represents a direct bond.

[0047] The spacer, or in the absence of a spacer, the targeting moiety, may be attached at the R4 and / or R5 residues in the compound of formula (II).

[0048] The linking group of R4-R5 is a reactive functional group such as a carboxylic acid or carboxyamide residue suitable for conjugating the dye to a targeting moiety by forming a chemical bond.

[0049] For example, when an amine-containing targeting moiety (T) is conjugated to a compound of formula (II), and R4 and / or R5 are alkyl substituted with a carboxylic acid, the carboxylic acid may be activated prior to conjugation through conversion to a more reactive form using an activating reagent, such as an N-hydroxysuccinimide (NHS) ester or mixed anhydride. The amine-containing targeting moiety is then treated with the resulting activated acid to form an amide bond to obtain the corresponding compound of formula (II). Typically, this reaction is carried out in an aqueous buffer (which may be cosolvent with DMSO or DMF (pH 8-9)) or in an organic solvent containing an organic base such as DIPEA, TEA, or NMM.

[0050] Alternatively, direct conjugation with an "unactivated" carboxylic acid can be performed.

[0051] Similarly, when the linking group at R4 and / or R5 is a carboxyamide group, the procedure for attachment of an appropriate targeting moiety is similar, although a linker activation step is generally not required and the dye and targeting moiety are processed directly.

[0052] The compounds of formula (I) or (II) above may have one or more asymmetric carbon atoms (alternatively referred to as chiral carbon atoms) and may therefore give rise to diastereomers and optical isomers. Unless otherwise indicated, the present invention further includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof.

[0053] As a non-limiting example, when the dyes of formula (I) or (II) are substituted with a D-glucamine group (Y is a C alkyl substituted with five hydroxy groups), the present invention includes the corresponding enantiomerically pure compounds as well as any of their stereoisomers, e.g., compounds with an L-glucamine group, or any possible mixtures of their D- / L-optical isomers.

[0054] The present invention further relates to compounds of the above formula (I) or (II) in which the functional groups of R1, R2 / R4 and / or R3 / R5, such as sulfonyl, carboxyamino or carboxylic acid groups, may be in the form of pharmaceutically acceptable salts.

[0055] In one embodiment, the present invention relates to a compound of formula (I) or (II), wherein Y is selected from linear or branched C1-C6 alkyl, cycloalkyl, and heterocyclyl substituted with 2 to 5 hydroxyl groups.

[0056] In a preferred embodiment, the present invention relates to compounds of formula (I) or (II), wherein Y is JPEG2026012682000003.jpg72166.

[0057] More preferably, the present invention relates to compounds of formula (I) or (II) wherein Y is a group of formula (ii) as defined above. Preferably, group (ii) has the following stereochemical configuration, obtained by using D-glucamine in the preparation of the compound: JPEG2026012682000004.jpg60166

[0058] Another embodiment of the present invention relates to compounds of formula (II), wherein m is 0 and the spacer S is represented by a direct bond, or m is 1 and the spacer is C-C 20 A hydrophilic moiety containing an alkyl, C3-C7 cycloalkyl, or aryl group. Preferably, the spacer is -(CH2) p COO-, -(CH2CH2O) p CH2CH2COO- and -(CH2CH2O) p CH2CH2NH-, where p is an integer between 0 and 20. Preferably, p is 2, 6 or 12.

[0059] In a further embodiment, T is a targeting moiety selected from a small molecule, a protein, a peptide, a peptidomimetic, an enzyme substrate, an antibody or any fragment thereof, and an aptamer.

[0060] Preferably, T is represented by a peptide, in particular α v β3, α v β5, α v Integrin receptors such as β6, α5β1, and preferably α v It is represented by the portion that interacts with the β3 integrin receptor.

[0061] In one preferred embodiment, R1 is a straight chain C4 alkyl substituted by -SO3H.

[0062] In another preferred embodiment, the present invention relates to compounds of formula (I) or (II), wherein Y represents group (ii) as defined above, otherwise represented by the following formula (Ia) or (IIa), respectively: JPEG2026012682000005.jpg104166 wherein R1, R2, R3, R4, R5 and X are as defined above.

[0063] Particularly preferred are the compounds of formula (I) listed in Table Ia and the compounds of formula (II) listed in Table Ib.

[0064] JPEG2026012682000006.jpg144166JPEG2026012682000007.jpg192166 JPEG2026012682000008.jpg153166JPEG2026012682000009.jpg137166JPEG2026012682000010.jpg206166JPEG2026012682000011.jpg88166

[0065] JPEG2026012682000012.jpg221166JPEG2026012682000013.jpg118166JPEG20260126820 00014.jpg207166JPEG2026012682000015.jpg150166JPEG2026012682000016.jpg201166

[0066] The present invention also relates to methods of synthesizing compounds of formula (I) or (II), which are near-infrared dyes that may be conjugated to a targeting moiety via a linking group, prepared as exemplified in the following description.

[0067] Thus, the present invention provides a compound of formula (I) or (II) as defined above for use as an optical imaging agent for diagnostic biomedical applications in mammals (humans and animals). Preferably, the mammalian subject to be imaged is a human.

[0068] In a preferred embodiment, the compounds of the invention are for use as imaging agents in the detection of normal (healthy) or abnormal (pathological) tissue, particularly tumors.

[0069] Preferably, the compounds of formula (I) or (II) as defined above are for use in the detection of normal (healthy) tissue using imaging techniques including, for example, angiography, perfusion imaging, biliary imaging and neuroimaging.

[0070] In a further preferred embodiment, the present invention provides a compound of formula (I) or (II) as defined above for use in detecting abnormal (pathological) tissues, such as primary tumor lesions, local or distant metastases, or preneoplastic lesions, particularly dysplasia and hyperplasia. In particular, the compound of formula (II) as defined above is preferably used in detecting and demarcating tumor margins in guiding surgical procedures for individual patients. A preferred use is when the tumor exhibits overexpression of a biological epitope selected from, for example, receptors present on the cell surface, enzymes, glycoproteins, lipid rafts, transmembrane proteins, and soluble factors present in serum, plasma, or interstitial space. Preferably, the biological epitope is an integrin receptor for vitronectin, fibrogen, and / or transforming growth factor-β (TGF-β).

[0071] The present invention also provides a compound of formula (I) or (II) for use as a fluorescent probe as defined above, wherein the detection and demarcation of tumors is carried out under NIR radiation. Preferably, such detection and demarcation of tumors is carried out before, during, or after a surgical procedure to remove such tumor tissue. Fluorescence-guided surgical procedures are an example of such use.

[0072] Furthermore, the present invention provides a compound of formula (I) or (II) as defined above for use in the detection of inflamed tissue, fibrotic tissue, ischemic tissue, or tissue with an abnormal metabolic rate.

[0073] The present invention also provides a method for imaging tissues and cells, comprising the steps of: i) contacting a cell or tissue with a compound of formula (I) and (II); ii) illuminating the tissue or cells with a wavelength absorbed by the imaging agent; iii) detecting the near-infrared emission using a fluorescence camera.

[0074] Preferably, the step of contacting cells or tissues with the imaging agent of formula (I) or (II) is accomplished by local or topical application (e.g., by spraying, dipping, or application of an ointment, foam, or cream), or by systemic application (enteral or parenteral administration).

[0075] The present invention further relates to a pharmaceutical diagnostic composition comprising a compound of formula (I) or a conjugate of formula (II) as defined above, and at least one pharmaceutically acceptable carrier or excipient.

[0076] In particular, the present invention relates to a pharmaceutical composition comprising a dye of formula (I), or a salt thereof, and one or more pharmaceutically acceptable adjuvants, excipients or diluents.

[0077] Alternatively, the present invention provides a compound wherein R4 and / or R5 are CONH-(S) as defined above. m The present invention relates to a pharmaceutical composition comprising a conjugate of formula (II), wherein -C1-C6 alkyl is substituted by -T, or a salt thereof, and one or more pharmaceutically acceptable adjuvants, excipients or diluents.

[0078] Another aspect of the present invention relates to a diagnostic kit comprising a compound of formula (I) or (II) as defined above. The kit may further comprise additional adjuvants for performing optical imaging. These adjuvants may be, for example, suitable buffers, containers, detection reagents, or instructions for use. The kit preferably includes all materials for intravenous administration of the compounds of the present invention.

[0079] The compounds of the present invention can be administered either systemically or locally to the organ or tissue to be imaged prior to the imaging procedure. For example, the compounds can be administered intravenously. In another embodiment, they can be administered parenterally or enterally.

[0080] The composition is administered in a dosage effective to obtain the desired optical image of the tumor, tissue or organ, which can vary widely depending on the compound used, the tissue subjected to the imaging procedure, the imaging equipment used, etc.

[0081] The exact concentration of the imaging agent depends on the experimental conditions and desired results, but can typically range from 0.000001 mM to 0.1 mM. The optimal concentration is determined by systematic variation until satisfactory results with minimal background fluorescence are obtained.

[0082] Once administered, the imaging agents of the present invention are exposed to light or other forms of energy capable of passing through tissue layers. Preferably, the wavelength or wavelength range of the radiation is comparable to the excitation wavelength or wavelength range of the photosensitizing agent and has low absorption by non-target cells and the rest of the subject (including blood proteins).

[0083] Typically, the optical signal is detectable by observation or instrumentation, and the response is related to fluorescence or light intensity, distribution and lifetime.

[0084] Synthesis Description The preparation of compounds of formula (I) or (II) (as such or in the form of a physiologically acceptable salt) is a further object of the present invention. The cyanine dyes and dye-conjugates of the present invention can be prepared, for example, according to the methods described in the following sections and experimental part.

[0085] General teachings regarding the preparation of cyanine dyes can be found in Mujumdar RB et al., Bioconjugate Chem. 1993, 4(2):105-111, on the synthesis and labeling of sulfoindocyanine dyes. However, the cyanines of the present invention feature a unique functionalization pattern not present in compounds of the art, which required the design of appropriate synthetic methods. Indeed, unlike other known cyanines, the compounds of the present invention possess three functional moieties (carboxylic acid or amide groups) that must be derivatized in different ways, thus necessitating the use of protecting groups in most cases to direct reactions at the desired functional groups.

[0086] Manipulating cyanines through the strong pH and temperature conditions required for protecting group removal is known to pose difficulties, as the stability of the cyclohexenyl-polymethine scaffold can be compromised, sometimes resulting in severe dye degradation.

[0087] Furthermore, when deprotecting the carboxylic acid groups of R1-R5, an additional obstacle may be encountered due to possible hydrolysis and decomposition of the amide group -CONH-Y (typically, amide derivatives can be hydrolyzed in concentrated alkaline media; see, for example, Yamana et al., Chem. Pharm. Bull., 1972, 20(5), 881-891).

[0088] In one preferred embodiment, the protecting group for the moiety R4 or R5 is an ester group. More preferably, an ethyl ester group can be advantageously used.

[0089] Preparation of cyanine dyes of formula (I) According to the present invention, compounds of formula (I) can be prepared by the general sequence of synthetic steps reported in Scheme 1 below.

[0090] JPEG2026012682000017.jpg204166

[0091] In the above scheme 1, R1, R2, R3, X and Y are as defined above, and Pg is absent or a suitable protecting group.

[0092] Thus, the process of the present invention comprises the following steps: a) treating an appropriate amount of 5-carboxy-2,3,3-trimethylindolenines of formula (III) and (IV) with a polyhydroxylated amine such as glucamine, meglumine, glucosamine, trometamol, serinol, or isoserinol, having a suitable protecting group on the hydroxy moiety; b) reacting intermediate (V) and intermediate (VI) obtained in step a) with 2-chloro-1-formyl-3-(hydroxymethylene)-1-cyclohexene to obtain a cyanine intermediate of formula (VII), wherein R, R, Y and Pg are as defined above; c) optionally, removing the protecting group (Pg) of the Y group from intermediate (VII); d) Displacing the chloro atom on intermediate (VII) with a suitable nucleophile to give the final product of formula (I) or a salt thereof.

[0093] According to step a), the reaction of polyhydroxylated amines with derivatives (III) and (IV) can be carried out in solvents such as dimethylformamide, dimethylacetamide, dimethyl sulfoxide, or acetonitrile for a suitable time ranging from 30 minutes to several hours at room temperature by activation of the carboxylate group with a coupling agent selected from HATU, TBTU, HBTU, PyBOP, DCC, DSC, and DCC-NHS, and an organic base such as TEA, DIPEA, NMM, or pyridine. This derivatization of the carboxylic acid can be carried out on alkylated indolenines or indoles prior to quaternization. In this case, it is important to protect the hydroxyl groups of the polyhydroxylated amines with appropriate protecting groups, such as acetyl, before alkylation with sultone or bromohexanoic acid. This alkylation can be carried out at elevated temperatures (e.g., 90°C-180°C) for several hours (typically 12 hours to 5 days) with the solution stirred, either neat or in a high-boiling solvent such as butyronitrile, sulfolane, 1,2-dichlorobenzene, dimethylacetamide, dimethylformamide, or dimethyl sulfoxide.

[0094] According to step b), the reaction can be carried out with the Vilsmeier reagent in the bis-anilide or bis-aldehyde form (reported in Scheme 1). The reaction can be carried out in various solvents, such as ethanol, methanol, acetic anhydride or acetic acid, with or without the addition of various bases, such as trimethylamine, pyridine, sodium acetate, potassium acetate, at various temperatures ranging from 45°C to 120°C, stirring the mixture for several hours (typically 2-24 hours).

[0095] According to step c), any protecting group of intermediate (VII) can be removed by the procedure described, for example, in TW Green and PG M Butts, Protective Groups in Organic Synthesis, Wiley, NY 2007, 4 thEd., Ch. 5. Unlike other classes of cyanines, these dyes showed greater stability at acidic pH and even at higher temperatures for several hours.

[0096] According to step d), the reaction can be carried out using various protocols depending on the X-R3 substituent. If a phenol or its derivative, e.g., phenol-SO3H, is introduced, the dye can be heated in DMSO in the presence of an inorganic base such as sodium or potassium carbonate. On the other hand, if the chloro is replaced by a phenyl or its derivative, the reaction can be carried out in degassed water or a mixture of degassed water and a cosolvent such as methanol or ethanol in the presence of a Pd catalyst such as Pd acetate or tetrakis Pd (which may also be a base such as sodium or potassium carbonate) and heated for a shorter period of time.

[0097] In the case where R1 has the same meaning as R2, only one reaction a) is carried out and the subsequent step b) is carried out with two units of intermediate (V) or (VI) instead of one unit of intermediate (V) and one unit of intermediate (VI).

[0098] Alternatively, when R1 has the same meaning as R2 and is a linear or branched C1-C6 alkyl substituted by -SO3H, the compounds of formula (I) can also be prepared according to Scheme 2 below: JPEG2026012682000018.jpg136166

[0099] In the above Scheme 2, R1 is a linear or branched C1-C6 alkyl substituted by -SO3H, and X, Y and R3 are as defined above.

[0100] Thus, another process of the present invention comprises the following steps: f) reacting at least two equivalent indolenine intermediates (III) (wherein R is a linear or branched C-C alkyl substituted by —SOH) with a Vilsmeier reagent (in the form of a bis-aldehyde or bis-anilide) to obtain the corresponding cyanine intermediate of formula (IX); g) displacing the chloro atom on intermediate (IX) with a suitable nucleophile to give intermediate (X); h) treating an appropriate amount of intermediate of formula (X) with a polyhydroxylated amine, such as glucamine, meglumine, glucosamine, trometamol, serinol or isoserinol, to obtain the final product of formula (I) or a salt thereof.

[0101] According to step f), the reaction can be carried out in various solvents such as, for example, ethanol, methanol, acetic anhydride or acetic acid, with or without the addition of various bases such as trimethylamine, pyridine, sodium acetate, potassium acetate, at various temperatures ranging from 45° C. to 120° C., by stirring the mixture for several hours (typically 2 to 24 hours).

[0102] According to step g), the reaction can be carried out using various protocols depending on the X-R3 substituent. If a phenol or its derivative, e.g., phenol-SO3H, is introduced, the dye can be heated in DMSO in the presence of an inorganic base such as sodium or potassium carbonate. On the other hand, if the chloro is replaced by a phenyl or its derivative, the reaction can be carried out in degassed water or a mixture of degassed water and a cosolvent such as methanol or ethanol in the presence of a Pd catalyst such as Pd acetate or tetrakis Pd (which may also be a base such as sodium or potassium carbonate) and heated for a shorter period of time.

[0103] According to step h), the reaction of the polyhydroxylated amine with the derivative (X) can be carried out in a solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, acetonitrile, for a suitable time ranging from 30 minutes to several hours, at room temperature, by activation of the carboxylate group with a coupling agent selected from, for example, HATU, TBTU, HBTU, PyBOP, DCC, DSC and DCC-NHS, and an organic base such as TEA, DIPEA, NMM or pyridine.

[0104] In a further embodiment, the compounds of formula (I) prepared according to the process of the present invention can be suitably converted into other compounds of formula (I) by operating according to the conditions of known synthesis, the following being examples of possible conversions: e) converting a compound of formula (I) where R2 is -COOH, i.e., a compound of formula (Ib), into the corresponding compound of formula (I) where R2 is -CONH2, i.e., a compound of formula (Ic): JPEG2026012682000019.jpg49166

[0105] According to step e), the conversion of the carboxylic acid of formula (Ib) into the corresponding carboxamide of formula (Ic) can be achieved in various methods and experimental conditions widely known in the art for the preparation of carboxamides. For example, the carboxylic acid can first be converted into a suitable activated ester, and then reacted with an ammonium salt such as NH4Cl, preferably in the presence of a coupling agent such as HBTU.

[0106] Preparation of the conjugate compound of formula (II) The cyanine derivative of formula (I), or a salt thereof, can be conjugated with a suitable targeting moiety (with or without a spacer) to give the corresponding compound of formula (II). Conjugation can be achieved according to various procedures known in the art, for example, via direct coupling of the carboxylic acid group of the compound with a nucleophilic residue of the targeting moiety (with or without a spacer), or by prior activation of the carboxylic acid group, whereby the carboxylic acid group is converted to a more reactive group, e.g., an ester such as NHS, prior to coupling.

[0107] In one embodiment, in the case of activation of a carboxylic acid via formation of an NHS ester, the present invention provides a dye of formula (XIa) JPEG2026012682000020.jpg81166 or a dye of formula (XIb) JPEG2026012682000021.jpg85166, During the ceremony, X is a direct bond or -O-; Y is a group selected from linear or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclyl substituted by at least two hydroxyl groups; R1 is a straight or branched C1-C6 alkyl substituted with a group selected from -SO3H, -COOH, -CONH2 and -COO-C1-C6 alkyl; and R3 is hydrogen, -SO3H, or a linear or branched C1-C6 alkyl substituted by -COOH or -CONH-Y, where Y is a group selected from linear or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclyl substituted by at least two hydroxyl groups; The method includes reacting a dye of formula (XIa) or (XIb) with a targeting moiety.

[0108] When the compounds of formula (I) or (II) prepared according to the above-described processes are obtained as mixtures of isomers, it is within the scope of the present invention to separate them into the single corresponding isomers of formula (I) or (II) using conventional techniques.

[0109] The final compounds may be isolated and purified using conventional procedures, such as chromatography and / or crystallization and salt formation.

[0110] The compounds of formula (I) or (II) as defined above can be converted into pharmaceutically acceptable salts. The compounds of formula (I) or (II) as defined above or their pharmaceutically acceptable salts can then be formulated with a pharmaceutically acceptable carrier or diluent to provide a pharmaceutical composition. [Example]

[0111] [Experimental Part] The invention and its specific embodiments described in the following parts are merely examples and are not to be considered as limitations of the invention, they are meant to show how the invention can be implemented and are meant to be illustrative without limiting the scope of the invention.

[0112] Materials and equipment All chemicals and solvents used in the reactions were reagent grade. Analytical grade solvents were used for chromatographic purification. Most of the reagents, including the targeting moiety, were commercially available products unless otherwise reported (e.g., panitumumab (Vectibix, Amgen; CASNr: 339177-26-3); c(RGDfK) (Cyclo(Arg-Gly-Asp-D-Phe-Lys), Bachem; CASNr: 161552-03-0).

[0113] All synthetic compounds were purified by reversed-phase chromatography (RP-HPLC) and characterized by mass spectrometry using an LC / MS instrument equipped with a UV-VIS detector and an ESI source. Analyses were performed on a Waters Atlantis dC18 5 μm, 4.6 × 150 mm column using a gradient of 10 mM CH3COONH4 in phase A and acetonitrile in phase B. The measured mass / charge ratio is listed for each compound.

[0114] The absorbance (Abs) of the compounds of the present invention was determined using a dual-beam UV-VIS spectrophotometer (Lambda 40, Perkin Elmer). Measurements of emission / excitation (Em / Ex) spectra and absolute fluorescence quantum yields (Φ) were performed using a spectrofluorometer (FluoroLog-3 1IHR-320, Horiba Jobin Yvon) equipped with an F-3018 integrating sphere accessory. Measurements were performed using excitation wavelengths at the absorbance maxima of the various dyes, and samples were excited with a 450 W xenon light source. Detection was performed using a photomultiplier tube (PMT-NIR) cooled detector or a TBX-04 detector. Dye solutions were carefully prepared to have absorbances below 0.1 (optical density) to minimize reabsorption phenomena.

[0115] In vivo imaging experiments were performed using an IVIS Spectrum In Vivo Imaging System (Perkin Elmer Inc.) equipped with 10 narrow-band excitation filters (30 nm bandwidth) and 18 narrow-band excitation filters (20 nm bandwidth) (range 430–850 nm).

[0116] JPEG2026012682000022.jpg176166

[0117] Abbreviations for individual amino acid residues are conventional: for example, Asp or D is aspartic acid, Gly or G is glycine, and Arg or R is arginine. Amino acids referred to herein should be understood to be in the L-isomer configuration unless otherwise indicated (e.g., the Phe or "f" in c(RGDfK) shown in the abbreviation list is the D-isomer form).

[0118] Example 1: Synthesis of Compound 11 Preparation of intermediate (Va) 5-Carboxy-2,3,3-trimethyl-1-(4-sulfobutyl)-3H-indol-1-ium (10.8 g, 31.9 mmol) was suspended in dry DMF (100 mL) under a N atmosphere; D-glucamine (6.9 g, 38.2 mmol), DIPEA (11 mL, 63.7 mmol), and HATU (14.5 g, 38.2 mmol) were added. The solution was stirred at RT for 16 h, and then cold diethyl ether (200 mL) was added. The mixture was filtered, and the solid was washed with ethyl acetate (2 × 50 mL). The solid was dissolved in water and purified by flash chromatography on a pre-packed C18 silica column using a 0.1% ammonium acetate / acetonitrile gradient. Fractions containing the pure product were combined, evaporated under vacuum, and lyophilized three times to give a pale pink solid (13.6 g, 80% yield), HPLC purity (270 nm): 94%. MS: [M+H] + 504.2.

[0119] Preparation of intermediate (VIIa) Acetic acid (8.65 mL) was added to a suspension of intermediate (Va) (2.0 g, 3.98 mmol) in acetic anhydride (10 mL). The mixture was heated at 45° C., and 2-chloro-1-formyl-3-(hydroxymethylene)-1-cyclohexene (377.8 mg, 2.19 mmol) was added, giving a yellow solution. The temperature was raised to 75° C. (the solution turned green-brown), and sodium acetate (408 mg, 4.97 mmol) was added, giving an immediate green solution. The solution was stirred at 100° C. for 3 h, then cooled to RT, and the solvent was removed under reduced pressure. The crude product was dissolved in water-acetonitrile and purified by flash chromatography on a pre-packed C18 silica column using a water-acetonitrile gradient. Fractions containing the pure product were combined and distilled under vacuum to give a green solid (2.5 g, 40% yield). HPLC purity (780 nm): 90%. MS: [M+H] + 1560.5.

[0120] Preparation of intermediate (VIIIa) Intermediate (VIIa) (2.5 g, 1.59 mmol) was dissolved in water / acetonitrile 1 / 1 (20 mL) and the pH was adjusted from 2.2 to 1.5 with 1N HCl. The solution was stirred at 80° C. for 16 hours. The crude product was purified by flash chromatography on a pre-packed C18 silica column using a water-acetonitrile gradient. Fractions containing the pure product were combined, evaporated under vacuum, and lyophilized to give a green solid (1.09 g, 60% yield). HPLC purity (780 nm) 95%. MS: [M+H] + 1140.7.

[0121] Synthesis of compound 11 To a solution of intermediate (VIIIa) (1.09 g, 0.95 mmol) in degassed water (20 mL) were added 4(2-carboxyethyl)-benzeneboronic acid (332 mg, 1.71 mmol), Pd(PPh3)4 (165 mg, 0.14 mmol), and sodium carbonate (181 mg, 1.71 mmol). The mixture was stirred at 80 °C for 16 h under a nitrogen atmosphere. Then, after cooling at RT, the pH was adjusted to 6.5 with 2 N HCl. The crude mixture was purified by flash chromatography on a pre-packed C18 silica column using a water-acetonitrile gradient. The fractions containing the pure product were combined, evaporated under vacuum, and lyophilized to give a green solid (0.714 g, 60% yield). HPLC purity (780 nm): 99%. MS: [M+H] + 1253.5.

[0122] Example 2: Synthesis of Compound 12 Compound 11 (56 mg, 0.0434 mmol) prepared in Example 1 was suspended in dry DMSO (5 mL) under a nitrogen atmosphere. NMM (19 μL, 0.174 mmol), HATU (66 mg, 0.174 mmol), and D-glucamine (39 mg, 0.217 mmol) were added. After stirring at RT for 2 h, the reaction mixture was precipitated in cold ethyl acetate (30 mL). The green solid was dissolved in water and purified by flash chromatography on a pre-packed C18 silica column using a 0.1% ammonium acetate-acetonitrile gradient. The product-containing fractions were combined, evaporated under vacuum, and lyophilized three times to give a green solid as the ammonium salt (69.88 mg). To remove the ammonium counterion, the solid was dissolved in water and loaded onto a C18 cartridge. Washed with water (2 CV), 0.1% HCOOH (2 CV), water (5 CV), and eluted with 1 / 1 water / acetonitrile. The solvent was distilled under vacuum and lyophilized to give a green solid (37 mg, 60% yield). HPLC purity (780 nm): 100%. MS: [M+H] + 1419.4.

[0123] Example 3: Synthesis of Compound 13 Preparation of intermediate (VIb) In a dry round-bottom flask, 2,3,3-trimethyl-3H-indole-5-carboxylic acid (711 mg, 3.5 mmol) was solubilized in dry DMF (4 mL) under a nitrogen atmosphere, followed by the addition of DIPEA (380 μL, 4.90 mmol). After 30 min of stirring at RT, a solution of TBTU (603 mg, 4.20 mmol) in dry DMF (2 mL) was added. After 1 h of stirring at RT, a suspension of D-glucamine (312 mg, 3.85 mmol) in dry DMF (2 mL) was added. After overnight, the reaction was not complete, so the same amounts of TBTU, DIPEA, and D-glucamine were added and stirred for an additional 2 h. The mixture was dried under vacuum and purified on a pre-packed C18 silica column using a water-acetonitrile gradient. The fractions containing the pure product were combined, evaporated under vacuum, and lyophilized to give a whitish-brown powder (888.2 mg, 70% yield). HPLC purity (270nm) 93% purity, MS: [M+H] + 502.57.

[0124] Acetic anhydride (3 mL) and pyridine (0.5 mL) were added to this intermediate (888.2 mg, 2.42 mmol) to give a suspension that gradually solubilized over time. The mixture was kept under stirring in a nitrogen atmosphere at RT for 4 hours. The solution was concentrated in vacuo and purified on a pre-packed C18 silica column using a water-acetonitrile gradient. The fractions containing the pure product were collected, concentrated in vacuo, and lyophilized to give a whitish-brown solid (861.4 mg, 62% yield). HPLC purity (270 nm) 97.5%, MS: [M+H] + 204.

[0125] In a round-bottom flask, the product thus obtained (1.103 g, 1.91 mmol) and 1-bromohexanoic acid (933 mg, 4.77 mmol) were solubilized in 1,2-dichlorobenzene (6 mL). The mixture was heated at 130 °C under a nitrogen atmosphere for 6 hours, then another 1-bromohexanoic acid (933 mg, 4.77 mmol) was added, and the reaction was maintained overnight under the same conditions. The crude product was washed with diethyl ether, the solvent was decanted, and the solid was dissolved in acetonitrile and purified on a pre-packed C18 silica column using a water-acetonitrile gradient. The product-containing fractions were collected and concentrated in vacuo to give a red oil (861.4 mg, 62% yield). HPLC purity (270 nm) 97.5%, MS: [M+H] + 691.0.

[0126] Preparation of intermediate (VIIb) In a dry round-bottom flask, intermediate (Va) (1.170 g, 2.33 mmol), prepared as in Example 1, and (VIb) (1.79 g, 2.33 mmol) were suspended in acetic anhydride (20 mL) and acetic acid (5 mL). The mixture was heated at 45° C. until the two powders were completely solubilized. 2-Chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (430 mg, 2.49 mmol) was then added, and the mixture was heated to 50° C. Potassium acetate (237 mg, 2.89 mmol) was added, and the mixture was heated at 100° C. for 2 hours. The solvent was removed in vacuo, and the crude green solid was purified on a pre-packed C18 silica column using a water-acetonitrile gradient. The product-containing fractions were collected, concentrated in vacuo, and lyophilized to give a green powder (1.221 g, 35% yield). HPLC purity (780nm) 72%, MS: [M+H] + 1540.6.

[0127] Preparation of intermediate (VIIIb) Intermediate (VIIb) (701 mg, 0.33 mmol) was solubilized in acetonitrile (3 mL) and water (15 mL) was added. The solution was acidified to pH 1.6 with 1N HCl and heated at 80° C. for 4 hours, then at 55° C. overnight. The organic solvent was removed under reduced pressure and the aqueous solution was purified on a pre-packed C18 silica column using a water-acetonitrile gradient. The fractions containing the product were collected, concentrated under vacuum, and lyophilized to give a green powder (179 mg, 50% yield). HPLC purity (780 nm) 95%, MS: [M+H] + 1120.3.

[0128] Synthesis of compound 13 In a dry round-bottom flask, intermediate (VIIIb) (115 mg, 1.03 mmol) was solubilized in degassed water (3 mL), followed by the addition of phenylboronic acid (22.7 mg, 1.85 mmol), sodium carbonate (19.6 mg, 1.85 mmol), and palladium tetrakis (17.8 mg, 0.15 mmol). The mixture was heated at 80°C for 2 hours under a nitrogen atmosphere. The solution was adjusted to pH 7.15 with 0.1 N HCl and purified on a pre-packed C18 silica column using a water-acetonitrile gradient. The product-containing fractions were collected, concentrated under vacuum, and lyophilized to give a green powder (99 mg, 83% yield). HPLC purity (780 nm) 98%, MS: [M+H] + 1162.2.

[0129] Example 4: Synthesis of Compound 14 A solution of intermediate (VIIIb) (120 mg, 0.107 mmol) prepared as in Example 2 in dry DMSO (5 mL) was added dropwise to a suspension of phenol (101 mg, 1.07 mmol) and anhydrous potassium carbonate (148 mg, 1.07 mmol) in dry DMSO (7 mL) under nitrogen. The mixture was stirred at 50 °C for 4 h. After cooling to RT, cold diethyl ether (30 mL) was added, and the solid was filtered and washed twice with cold diethyl ether. It was dissolved in water, and the pH was adjusted from 11.5 to 6 with 0.5 N HCl. The crude solid was purified by flash chromatography on a pre-packed C18 silica column using a 0.1% ammonium acetate-acetonitrile gradient. Fractions containing the pure product were combined and distilled under vacuum. To remove the ammonium counterion, the pH was adjusted to 1.6, and the product was loaded onto a C18 silica cartridge, washed with water, and eluted with 1:1 water-acetonitrile. The solvent was distilled under vacuum and the aqueous solution was lyophilized to give a green solid (42 mg, 33% yield). HPLC purity (780 nm) 99.3%. MS: [M+H] + 1178.3.

[0130] Example 5: Synthesis of Compound 15 A solution of intermediate (VIIIb) (20 mg, 0.018 mmol) prepared as in Example 2 in dry DMSO (3 mL) was added dropwise to a suspension of 4-hydroxybenzenesodium sulfonate (35 mg, 0.18 mmol) and anhydrous potassium carbonate (25 mg, 0.18 mmol) in dry DMSO (5 mL) under a nitrogen atmosphere. The mixture was stirred at 50° C. for 4 days. Cold diethyl ether (30 mL) was added to the brown mixture, and the solid was filtered and washed twice with cold diethyl ether. The solid was dissolved in water, and the pH was adjusted from 11.5 to 6 with 0.5 N HCl. The solution became green again. The crude product was purified by flash chromatography on a pre-packed C18 silica column using a water-acetonitrile gradient. The fractions containing the pure product were combined, evaporated under vacuum, and lyophilized to give a green solid (15 mg, 65% yield). HPLC purity (780 nm) 100%. MS: [M+H] + 1257.3.

[0131] Example 6: Synthesis of Compound 16 A solution of intermediate (VIIIa) (30 mg, 0.026 mmol) prepared as in Example 1 in dry DMSO (3 mL) was added dropwise to a suspension of 4-hydroxybenzenesodium sulfonate (14 mg, 0.08 mmol) and anhydrous potassium carbonate (10 mg, 0.08 mmol) in dry DMSO (3 mL) under a nitrogen atmosphere. The mixture was stirred at 80°C for 6 hours. Chilled ethyl acetate (20 mL) was added to the brown mixture, and the solid was filtered, dissolved in water, and the pH was adjusted from 11.5 to 3 with 0.5 N HCl. The solution became green again. The crude product was purified by flash chromatography on a pre-packed C18 silica column using a water-acetonitrile gradient. The fractions containing the pure product were combined, evaporated under vacuum, and lyophilized to give a green solid (14 mg, 42% yield). HPLC purity (780 nm) 97.8%. MS: [M+H] + 1277.3.

[0132] Example 7: Synthesis of Compound 17 In a dry round-bottom flask, intermediate (VIIIa) (9 mg, 0.008 mmol) prepared as in Example 1 was solubilized in degassed water (2 mL), followed by the addition of phenylboronic acid (2.2 mg, 0.018 mmol) and palladium acetate (0.15 mg, 0.0006 mmol). The mixture was refluxed under nitrogen for 2 hours. The solution was purified on a pre-packed C18 silica column using a water-acetonitrile gradient. The product-containing fractions were collected, concentrated under vacuum, and lyophilized to give a green powder (7 mg, 75% yield). HPLC purity (780 nm) 98%, MS: [M+H] + 1184.3.

[0133] Example 8: Synthesis of Compound 19 Preparation of intermediate (IXa) Compound (IIIa) (180 mg, 0.53 mmol), N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline monohydrochloride (101 mg, 0.265 mmol), and sodium acetate (109 mg, 1.32 mmol) were dissolved in absolute ethanol (55 mL), and the solution was refluxed for 26 h. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on a silica C18 column using a water / acetonitrile gradient. Fractions containing the pure product were combined, evaporated under vacuum, and lyophilized three times to give a green solid (154 mg, 72% yield). HPLC purity (780 nm) 97%. MS: [M+H] + 815.2.

[0134] Preparation of intermediate (Xa) A solution of intermediate (IXa) (40 mg, 0.05 mmol) in dry DMSO (4 mL) was added dropwise to a suspension of phenol (45 mg, 0.49 mmol) and anhydrous potassium carbonate (68 mg, 0.49 mmol) in dry DMSO (8 mL) under a nitrogen atmosphere. The mixture was stirred at 50 °C for 8 h. After cooling to RT, cold diethyl ether (30 mL) was added, and the solid was filtered and washed twice with cold diethyl ether. It was dissolved in water, and the pH was adjusted from 12 to 6 with 0.5 N HCl. The crude solid was purified by flash chromatography on a pre-packed C18 silica column using a water / acetonitrile gradient. Fractions containing the pure product were combined and distilled under vacuum. The solvent was evaporated under reduced pressure, and the aqueous solution was lyophilized to give a green solid (26 mg, 61% yield). HPLC purity (780 nm) 98%. MS: [M+H] + 872.1

[0135] Synthesis of compound 19 TBTU (5.1 mg, 0.015 mmol) was added to a solution of intermediate (Xa) (3.3 mg, 0.0038 mmol) and DIPEA (6 μL, 0.031 mmol) in anhydrous DMF (5 mL). The solution was stirred at RT for 30 min, then serinol (1 mg, 0.011 mmol) was added and the solution was stirred for 90 min. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on a silica C18 column eluted with a water-acetonitrile gradient. Fractions containing the pure product were combined, concentrated under reduced pressure and lyophilized to give a dark green solid (2 mg, 53% yield). HPLC purity (780 nm) 98.5%. MS: [M+H] + 1019.1.

[0136] Similarly, compounds 18, 20, 21, 22 and 23 were prepared as described above following similar procedures but using different hydroxylated amines (as Y groups) or starting from scaffolds bearing phenyl instead of phenol.

[0137] Example 9: Synthesis of Compound 1 Compound 11 (5 mg, 0.004 mmol) was dissolved in dry DMF (3 mL) under an inert atmosphere. DIPEA (6.8 μL, 0.04 mmol) and TSTU (12 mg, 0.04 mmol) were added, and the solution was stirred at RT for 48 h. Then, cold diethyl ether (20 mL) was added, and the precipitate was filtered and washed twice with cold diethyl ether to give a green solid. HPLC purity (780 nm): 90%. MS: [M+H] + 1351.

[0138] The NHS ester was dissolved in a solution of c(RGDfK) (2.7 mg, 0.004 mmol) in 1 mL of borate buffer (pH 9). The reaction was stirred at RT for 24 h, and then the product was precipitated in cold diethyl ether and washed twice with cold diethyl ether to give a green solid. The crude solid was purified by HPLC chromatography on a Kromasil C8 column using a linear gradient of 0.1% ammonium acetate / acetonitrile. Fractions containing the pure product were combined, evaporated under vacuum, and lyophilized three times to give a green solid (3.9 mg, 50% yield). HPLC purity (780 nm): 97.6%. MS: [M+H] + June 1840.

[0139] Example 10: Synthesis of Compound 3 [Procedure A via direct coupling] In a dry round-bottom flask, compound 13 (2 mg, 0.0017 mmol) was solubilized in DMF (1 mL) containing DIPEA (0.6 μl, 0.003 mmol) under a nitrogen stream at RT. After 30 min of stirring at RT, a solution of TBTU (0.6 mg, 0.002 mmol) in dry DMF (1 mL) was added. After 1 h of stirring at RT, a solution of c(RGDfK) (1.4 mg, 0.002 mmol) in dry DMF (1 mL) was added dropwise to the stirred solution. The reaction was stirred overnight at RT, then the solvent was removed in vacuo, and the crude product was purified on an analytical HPLC C18 silica column using a 0.1% ammonium acetate-acetonitrile gradient. Fractions containing the pure product were combined, concentrated in vacuo, and lyophilized three times to give a green powder (0.7 mg, 23% yield). HPLC purity (780nm) 98%, MS: [M / 2] + 872.8.

[0140] [NHS ester-mediated procedure B] In a dry round-bottom flask, compound 13 (5 mg, 0.0043 mmol) was solubilized in dry DMF (2 mL) containing DIPEA (2.2 μl, 0.0129 mmol), and a solution of TSTU (3.78 mg, 0.0129 mmol) in dry DMF (1 mL) was added at room temperature under a nitrogen stream. The green solution was stirred overnight at room temperature, and then the product was precipitated by the addition of cold diethyl ether. The solid was centrifuged, washed twice with diethyl ether, and used in the following step without further purification. The NHS ester was dissolved in a solution of c(RGDfK) (2.2 mg, 0.0043 mmol) in borate buffer (pH 9) (1 mL), and the solution was stirred overnight at room temperature. The pH was then adjusted to 7 with 0.1 N HCl, and the crude product was purified on an analytical HPLC C18 silica column using a 0.1% ammonium acetate-acetonitrile gradient. The fractions containing pure product were combined, concentrated in vacuo, and lyophilized three times to give a green powder (3.9 mg, 52% yield). HPLC purity (780 nm) 97.7%, MS: [M / 2] + 872.8.

[0141] Example 11: Synthesis of Compound 2 Compound 15 (6 mg, 0.0048 mmol) was suspended in dry DMF (6 mL), and then NMM (2.6 μL, 0.024 mmol) and TSTU (7.2 mg, 0.024 mmol) were added. The solution was stirred at RT for 16 h. Chilled diethyl ether (30 mL) was added, and the precipitate was filtered and washed twice with chilled diethyl ether. The green solid was used in the following step without further purification. HPLC (780 nm): 88%, MS: [M+H] + 1354.3.

[0142] The NHS ester was dissolved in borate buffer (pH 9) (1 mL) and a solution of c(RGDfK) (3.4 mg, 0.0048 mmol) in borate buffer (pH 9) (1 mL) was added. The solution was stirred at RT for 5 h, then the pH was adjusted to 6.5 with 0.1 N HCl, and the crude product was purified by HPLC chromatography on a phenyl-C18 column using a linear gradient of 0.1% ammonium acetate-acetonitrile. Fractions containing the pure product were combined, distilled under vacuum, and lyophilized three times to give a green solid (6.4 mg, 71% yield). HPLC purity (780 nm) 99.6%. MS: [M+H] + May 1845.

[0143] Example 12: Synthesis of Compound 4 Compound 14 (12 mg, 0.0102 mmol) was dissolved in dry DMF (1 mL) under a nitrogen atmosphere. DIPEA (4.8 μL, 0.0286 mmol) and TBTU (4.6 mg, 0.0143 mmol) were added, and after stirring at RT for 1 h, c(RGDfK) (6.15 mg, 0.0102 mmol) was added. The reaction was stirred at RT for 16 h, then cold diethyl ether (25 mL) was added, and the precipitate was filtered and washed twice with cold diethyl ether. The crude solid was dissolved in water and purified by HPLC chromatography on a phenyl-C18 column using a linear gradient of 0.1% ammonium acetate-acetonitrile. Fractions containing the pure product were combined, evaporated under vacuum, and lyophilized three times to give a green solid (12.3 mg, 67% yield). HPLC purity (780 nm) 98.3%, MS: [M+H] + 1763.6.

[0144] Example 13: Synthesis of Compound 5 The monoclonal antibody EGFR ligand panitumumab (6 mg) was diluted to 5 mg / mL in PBS, and the pH was adjusted by adding 120 μL of 1.0 M potassium phosphate (pH 9). Compound 15-NHS ester (prepared as described for Compound 2 in Example 11) was dissolved in DMSO at a concentration of 10 mg / mL; the dye and antibody were then immediately mixed in a 2.5:1 molar ratio and kept in the dark at room temperature for 3 hours. After 3 hours, the conjugation reaction mixture was layered onto a Zeba Spin column equilibrated with phosphate-buffered saline (PBS) and centrifuged at 1500 g for 2 minutes to separate the conjugate from the free dye. After filtration through a 0.22 μm polyethersulfone (PES) membrane, the conjugated panitumumab solution in PBS (pH 7.4) was analyzed by SE-HPLC, RP-HPLC, and UV / VIS spectrophotometry to determine concentration and purity. The molar conjugate ratio (dye molecules bound per antibody) was 1.53.

[0145] Example 14: Synthesis of Compound 6 The small molecule CAIX ligand 4a (described in Wichert et al., Nat Chem 2015, 7, 241-249) was prepared and conjugated to compound 15 according to the published procedure. 11 mg of compound 15 (8.7 μmol) was dissolved in 1 mL of DMF, and then 4.5 mg of PyBOP (8.7 μmol) and 6 μL of DIPEA (35.0 μmol) were added under continuous stirring. After 20 min, 8 mg of small molecule 4a (13.0 μmol) was dissolved in 1 mL of DMF and added to the reaction mixture, which was stirred for an additional 30 min at room temperature. Purification by preparative HPLC was performed in 50% yield. The isolated pure product was characterized by HPLC-UV-VIS-MS-ESI(+) using a Waters Atlantis dC18 column (μm, 4.6 × 150 mm). HPLC purity (779nm):99%;MS:[M / 2] + 929.7.

[0146] Example 15: Synthesis of Compound 7 The small molecule CAIX ligand 8a (described in Wichert et al., Nat Chem 2015, 7, 241-249) was prepared according to the published procedure and conjugated to compound 15. 9 mg of compound 15 (7.1 μmol) was dissolved in 1 mL of DMF, and then 3.7 mg of PyBOP (7.1 μmol) and 5 μL of DIPEA (28.0 μmol) were added under continuous stirring. After 20 min, 11 mg of molecule 8a (11.0 μmol) was dissolved in 1 mL of DMF and added to the reaction mixture, which was stirred for an additional 30 min at RT. Purification by preparative HPLC was performed in 50% yield. The isolated pure product was characterized by HPLC-UV-VIS-MS-ESI(+) using a Waters Atlantis dC18 column (μm, 4.6 x 150 mm). HPLC purity (780nm):99%;MS:[M / 2] + 1157.8.

[0147] Example 16: Synthesis of Compound 8 9.9 mg of compound 15 (7.9 μmol) was dissolved in 3 mL of dry DMF, then 2 μL of NMM (18.2 μmol) and 7.11 mg of TSTU (23.6 μmol) were added, and the mixture was stirred at RT for 2 hours. The NHS ester of compound 15 (HPLC conversion 85.9%) was precipitated in 25 mL of ice-cold ethyl acetate. The precipitate was washed with ethyl acetate and dried under a stream of N2.

[0148] The NHS ester of compound 15 was dissolved in 1 mL of dry DMF. A solution prepared by dissolving 5.74 mg of EuK TFA salt (13.43 μmol) in 1 mL of DMF was added dropwise. Then, a solution of 13.72 μL of DIPEA (7.8 μmol) in 1 mL of DMF was added dropwise. The solution was stirred overnight at RT under a N2 atmosphere. The product (HPLC conversion 84%) was precipitated in 25 mL of ice-cold diethyl ether and purified on a pre-packed silica C18 column (BIOTAGE® SNAP ULTRA 26 g) equipped with an automated flash chromatography system (Combiflash Rf+) eluting with a water / acetonitrile gradient. Fractions containing the desired pure product were combined, concentrated under vacuum, and lyophilized to recover 6.65 mg of a green solid (HPLC purity area %: 98.7% (785 nm) and 100% (254 nm); [MH] + 1558.7). The yield from compound 15 was 54.0%.

[0149] Example 17: Synthesis of Compound 9 9.9 mg of compound 15 (7.9 μmol) was dissolved in 3 mL of dry DMF. 2 μL of NMM (18.2 μmol) and 7.11 mg of TSTU (23.6 μmol) were added, and the mixture was stirred at room temperature for 2 hours. The NHS ester of compound 15 (HPLC conversion 85.9%) was precipitated in 25 mL of ice-cold ethyl acetate. The precipitate was washed with ethyl acetate and dried under a stream of N2.

[0150] The NHS ester of compound 15 was dissolved in dry DMF (1 mL). A solution of EuK-(3-(2-naphthyl)-alanine)-tranexamic acid TFA salt (7.23 mg, 0.00945 mmol) prepared as described by Benesova et al., J Nucl Med 2015, 56:914-920, in 1 mL of DMF was added dropwise. Then, a solution of DIPEA (6.86 μL, 0.039 mmol) in 1 mL of DMF was added dropwise. The solution was stirred overnight at RT under a N2 atmosphere. The product (HPLC conversion 93%) was precipitated in 25 mL of ice-cold diethyl ether and purified on a prepacked silica C18 column (BIOTAGE® SNAP ULTRA 26 g) equipped with an automated flash chromatography system (Combiflash Rf+) eluting with a water / acetonitrile gradient. Fractions containing the desired pure product were combined, concentrated under vacuum, and lyophilized to recover 4.25 mg of a green solid (HPLC purity area %: 99.6% (785 nm) and 98% (254 nm); [MH] + The yield from compound 15 was 28.0%.

[0151] Example 18: Optical properties The compounds of the present invention were characterized in terms of their in vitro optical properties in aqueous media (i.e., water / PBS pH 7.4) and in clinical chemistry control serum (Seronorm, Sero SA) that mimics the chemical composition and optical properties of human serum. All dye or dye conjugate solutions were freshly prepared. ICG and S0456 were used as commercial references.

[0152] In particular, the excitation and emission maxima and absolute fluorescence quantum yields (Φ) of representative compounds of formula (I) and conjugates of formula (II) are shown in Table II.

[0153] JPEG2026012682000023.jpg137166

[0154] The compounds of the present invention are characterized by an absorption maximum within the range of about 760 nm to 810 nm. The dyes have fluorescence emission in the near-infrared region and high fluorescence quantum yields, even when conjugated to targeting moieties. In general, the dyes and conjugates exhibit higher fluorescence quantum yields than ICG and S0456.

[0155] Example 19: Affinity of human albumin (HSA) The binding affinity of the compounds of the present invention to human albumin was analyzed, and the results were compared with those of ICG and S0456 as references. The binding affinity to human serum albumin (HSA; Sigma Aldrich, A9511) was measured using two methods according to the level of binding affinity of the compounds.

[0156] The first method is optimal for compounds that strongly interact with HSA and is based on the analysis of the shift in the absorbance spectrum peak after incubation of the dye in a solution containing HSA. Briefly, samples are diluted with HSA at a fixed concentration (1 μM) in phosphate buffer for 5 min at 25 °C in a spectrophotometer before measurement. -6 ~4×10 -4 M) and measurements were taken at the wavelength of maximum absorbance of the shifted peak.

[0157] The second method is appropriate for compounds with low affinity for HSA and is based on measuring the absorbance variation of solutions containing a dye and various concentrations of HSA after ultrafiltration. Briefly, each compound is diluted in phosphate buffer at a fixed concentration (2 μM) with HSA dilutions (1 × 10 -6 ~4×10 -4 The samples were centrifuged (10,000 g, 25°C for 30 min) in a Microcon device (10 kDa MWCO, Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane (Millipore)), and absorbance measurements of the filtrate were obtained using a spectrophotometer at the wavelength of maximum absorbance of the fluorophore.

[0158] For both methods, the affinity constant (K A , M -1 ) was calculated by fitting the raw data with the following equation: JPEG2026012682000024.jpg32166In formula, ΔA / b = measured absorbance (b = 1 cm) K RL = K calculated by regression analysis A (curve fitting) Δε·Rt calculated by regression analysis (curve fitting) [L] = albumin concentration

[0159] In the first method, ΔA / b corresponds to the absorbance measured for each sample, while in the second method, ΔA / b is obtained by subtracting the absorbance of the control sample (dye without HSA) from the absorbance of each sample.

[0160] Both methods are similar to the first method (HSA K A =215,000M -1 ) and the second method (HSA K A =216,000M -1 ) was shown to provide comparable results as shown by parallel experiments performed with the commercially available cyanine dye IRDye 800CW carboxylate (LI-COR Inc., Lincoln, USA). However, the determination of affinity constants was more accurate when using the appropriate method as a function of the affinity level of the compound.

[0161] The binding affinity results measured for representative compounds of the invention using one of the two methods are reported in Table III and compared with the results obtained for the cyanine dye IRDye 800CW carboxylate dye as a reference compound.

[0162] JPEG2026012682000025.jpg115166

[0163] As shown in Table III, both the dyes and dye-conjugates of the present invention exhibited significantly lower binding affinities for human albumin compared to the known near-infrared dyes ICG and S0456, with affinity constants one or two orders of magnitude lower.

[0164] This advantageous feature is preserved in the dyes of the present invention even when conjugated to targeting moieties (e.g., compounds 2 and 4), since conjugation of the targeting moiety to the dye does not affect the affinity for human albumin.

[0165] Example 20: Receptor binding affinity The binding affinity of the conjugates of formula (II) to specific receptors was determined to assess whether the targeting efficacy of the molecular vectors was preserved after labeling with the dyes of the present invention.

[0166] As examples of small molecule and peptide / peptidomimetic conjugates, the receptor affinities of representative integrin-binding conjugates were determined using their IC 50 The half-maximal inhibitory concentration (HAI) was assessed by calculation using enzyme-linked immunosorbent assay (ELISA) as previously reported (Kapp et al., Sci. Rep. 2017, 7, 39805).

[0167] Briefly, a 96-well ELISA plate was coated overnight at 4°C with the extracellular matrix (ECM) protein vitronectin in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Each well was then washed with PBS-T-buffer (phosphate-buffered saline / Tween 20, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.01% Tween 20, pH 7.4) and blocked with TS-B-buffer (Tris-salts / BSA buffer; 20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, pH 7.5, 1% BSA) for 1 hour at RT. Meanwhile, dilution series of compounds and internal standards were prepared in an extra plate. After washing the assay plate three times with PBS-T, 50 μL of serial dilutions of human recombinant integrin α in TS-B-buffer were transferred to each well. v 50 μL of a solution of β3 (R&D Systems, 1 μg / mL) was transferred to the wells and incubated for 1 hour. The plate was washed three times with PBS-T buffer, and then the primary antibody anti-α v β3 was added to the plate. After incubation and washing three times with PBS-T, a secondary anti-IgG peroxidase-conjugated antibody was added to the plate and incubated for 1 hour. After washing the plate three times with PBS-T, the plate was developed by the rapid addition of 3,3',5,5'-tetramethylbenzidine (TMB) and incubated for 5 minutes in the dark. The reaction was stopped by the addition of 3M H2SO4, and the absorbance was measured at 450 nm using a plate reader (Victor3, Perkin Elmer).

[0168] IC of representative compounds 1, 2 and 4 50 were tested in duplicate and the resulting inhibition curves were analyzed using GraphPad Prism version 4.0 for Windows (GraphPad Software). The inflection points were determined as IC 50 Values ​​are defined. All experiments were performed using c(RGDfK) as an internal standard.

[0169] The tested molecular probes conjugated to c(RGDfK) were found to be human α v It showed comparable affinity for the β3 receptor and similar affinity to the unconjugated reference peptidomimetic c(RGDfK).

[0170] JPEG2026012682000026.jpg62166

[0171] Example 21: Cellular uptake The human melanoma cell line WM-266-4 (ATCC, CRL-1676) was used as an in vitro model to evaluate the cellular uptake of representative integrin-binding compounds 1, 2, and 4. (These cells bind to integrin receptors on the membrane, particularly α v Based on high expression of β3 (Capasso et al., PlosOne 2014).

[0172] Adherent cells at approximately 70% confluence were incubated with compound 1 or 3 (1 μM) for 2 h at 37 °C (5% CO) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin. After two washing steps with PBS, cells were detached using 0.1 mM EDTA in PBS, centrifuged, and suspended in buffer (PBS, 0.5% BSA, 0.1% NaN) for flow cytometry experiments. Fluorescence-activated cell sorting (FACS) was used to detect intracellular fluorescent signals as a measure of cellular uptake. Samples were excited with an argon laser, and emission was detected using a 670 nm long-pass filter. Fluorescence intensity values ​​were obtained from histogram statistics generated by the instrument software.

[0173] Specificity of receptor-mediated cellular uptakeTo assess the internalization of RGDfK, we performed experiments by incubating cells with the molecular probe in the presence of a high concentration (100 μM) of unlabeled molecular vector c(RGDfK) as a competitor. Residual internalization was calculated by setting the fluorescence intensity value in the absence of competitor as 100%.

[0174] moreover, Effect of biological fluids on cellular uptake To assess the uptake potential of compounds of the present invention, parallel experiments were performed in which cells were incubated with compounds of the present invention in the presence of human serum (Sigma Aldrich, H4522) derived from male AB plasma. Residual internalization was calculated by setting the fluorescence intensity value in the absence of serum at 100%. Such uptake assessment also represents an indication of the percentage of compound sequestered by plasma proteins as it diffuses through the vascular compartment before reaching the tissue of interest and specific target receptors. Table V shows the cellular uptake potential of representative compounds 2 and 4 of the present invention.

[0175] The compound exhibited high cellular uptake in the presence of human serum. Thus, it was observed that the cellular internalization of the compound is receptor-mediated and only slightly affected by binding to human serum proteins, particularly albumin (approximately 10-20% of the remaining uptake), confirming the compound's moderate to low binding affinity to human albumin (K, as shown in Example 10). A = about 1~6×10 3 M -1 Furthermore, the compounds of the present invention were compared with the reference compound ICG-RGD (Capozza et al., Photoacoustic 2018, 11, 36-45) and ICG-c(RGDfK), which was prepared in the same manner as ICG-RGD. These results show that, when incubated in the presence of human serum, the compounds of the present invention were surprisingly found to confer higher efficacy in cellular internalization than similar compounds known in the art.

[0176] JPEG2026012682000027.jpg62166

[0177] Remarkably, neither the interaction of the compounds with receptors on the cell surface nor the internalization of the receptor-probe complex within the cells was impaired by the conjugate dye structure, and in particular by the presence of a highly hydrophilic and highly sterically hindered moiety at position Y of the compound. Thus, the presence of a hydrophilic moiety on the conjugate dye confers highly efficient and specific receptor binding and probe internalization, even in the presence of plasma proteins that sequester conjugates lacking the hydrophilic moiety and negatively affect binding efficiency.

[0178] Example 22: Tumor uptake in animal models Human glioblastoma Tumor uptake experiments were performed using integrin receptors, particularly α v This study was conducted in an animal model of human glioblastoma (subcutaneously) overexpressing β3. Briefly, human glioblastoma U87MG cells (ATCC, HTB-14) were cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin. Male Balb / c nu / nu mice, 4–6 weeks old (Charles River Laboratories), were subcutaneously implanted (right flank) with approximately 10 million cells suspended in 0.1 mL of EMEM. Mice were housed four per cage with food and water available ad libitum. Animals were fed VRF1(P) sterile diet (Special Diets Services Ltd) until the end of the acclimation period (5 days). AIN-76a rodent chow (irradiated), a specialized diet that reduces autofluorescence, was then used until the end of the experiment. Tumor growth was 300-600 mm 3 The tumors were monitored by longitudinal assessment using calipers until the target size reached 3-4 weeks after cell transplantation. Imaging experiments were performed using a preclinical optical system, IVIS Spectrum (Perkin Elmer).

[0179] In vivo imaging was performed under gas anesthesia (6-8% Sevofluorane in oxygen). Animals were intravenously injected with the compound (lateral tail vein) and euthanized 24 hours after administration. Regions of interest (ROIs) were drawn in the excised tumor and healthy muscle tissue, and signal intensity (expressed as mean radiant efficiency) was assessed. The ratio between the fluorescent signal in the tumor and the muscle (background tissue) was then calculated to assess contrast.

[0180] Representative tumor-to-background ratios for compounds 1 and 4 are shown in Table VI. These results demonstrate significantly high tumor uptake, suggesting tumor-specific accumulation.

[0181] JPEG2026012682000028.jpg48166

[0182] Human head and neck cancer Tumor uptake experiments were performed, particularly with integrin receptor α v An animal model of human head and neck cancer (orthotopic) was performed using Detroit-562 cells overexpressing β6. Briefly, human pharyngeal carcinoma cells Detroit-562 (ATCC, CCL-138) were cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin. Male Balb / c nu / nu mice, 4–6 weeks old (Charles River Laboratories), were orthotopically implanted with approximately 2.5 million cells suspended in 0.03 mL of di-EMEM into the anterior tongue. Mice were housed four per cage with food and water available ad libitum. Animals were fed VRF1(P) sterile diet (Special Diets Services Ltd) until the end of the acclimation period (5 days). Then, AIN-76a rodent chow (irradiated) (Research Diets), a special diet that reduces autofluorescence, was used until the end of the experiment. Tumor growth was controlled at 10-20 mm. 3 The tumors were monitored by longitudinal assessment using calipers until they reached the target size (7-10 days after cell transplantation).

[0183] Imaging experiments were performed using a preclinical optical system, IVIS Spectrum (Perkin Elmer). Animals were intravenously injected (lateral tail vein) at 3 nmol / mouse, and 24 hours after administration, they were euthanized by anesthesia overdose, and their tongues were removed for ex vivo optical imaging. Regions of interest (ROIs) were set in the anterior (site of tumor cell implantation) and posterior (healthy tissue) regions of the tongue, and tumor-to-background ratios were obtained.

[0184] Ex vivo imaging performed 24 hours after administration of Compounds 1 and 2 revealed bright areas at the site of the tongue where tumor cells were implanted. In contrast, healthy areas at the back of the tongue showed low signal, suggesting low retention in healthy tissue. Administration of the compounds of the present invention revealed tumor location with moderate (TBR approx. 2) tumor-to-background contrast, as shown in Table VII.

[0185] JPEG2026012682000029.jpg64166

[0186] Human colorectal cancer Tumor uptake experiments were performed in an animal model of human colorectal cancer (subcutaneous) using HT-29 cells, which express low levels of integrin receptors. Briefly, human colorectal adenocarcinoma cells HT-29 (ATCC, HTB-38) were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin. Male athymic nude mice, 4-6 weeks old (Envigo), were subcutaneously implanted (right flank) with approximately 5 million cells suspended in 0.1 mL of serum-free medium. Mice were housed four per cage with food and water provided ad libitum. Animals were fed VRF1(P) sterile diet (Special Diets Services Ltd) until the end of the acclimation period (5 days). Then, AIN-76a rodent chow (irradiated) (Research Diets), a special diet that reduces autofluorescence, was used until the end of the experiment. Tumor growth was monitored at 300-600 mm. 3The tumor size was monitored by longitudinal evaluation using calipers until the tumor reached the target size (3-4 weeks after cell transplantation). Imaging experiments were performed using a preclinical optical system, IVIS Spectrum (Perkin Elmer).

[0187] In vivo imaging was performed under gas anesthesia (6-8% Sevofluorane in oxygen). Animals were intravenously injected with the compound of interest (lateral tail vein) and euthanized 24 hours after administration. Regions of interest (ROIs) were drawn in the excised tumor and healthy reference tissue (muscle), and signal intensity was assessed (expressed as mean radiant efficiency). The ratio between the fluorescent signal in the tumor and in the background tissue (muscle) was then calculated to assess the tumor-to-background ratio (TBR).

[0188] As shown in Table VIII, representative compounds 1 and 2 exhibited moderate tumor-to-background ratios (TBR approx. 4), allowing clear delineation of tumor tissue from the healthy background.

[0189] JPEG2026012682000030.jpg58166

[0190] JPEG2026012682000031.jpg152166

Claims

1. A compound of formula (I) During the ceremony, X is a direct bond or —O—; Y is a linear or branched C substituted with at least two hydroxyl groups. １ -C ６ Alkyl, C ３ -C ７ is a group selected from cycloalkyl and heterocyclyl; R1 and R2 each independently represent -SO ３ H, -COOH, -CONH ２ and -COO-C １ -C ６ a straight or branched C substituted with a group selected from alkyl １ -C ６ is alkyl; and R3 is hydrogen, —SO ３ H, or straight or branched C substituted by —COOH or —CONH—Y １ -C ６ alkyl, wherein Y is a linear or branched C alkyl substituted with at least two hydroxyl groups. １ -C ６ Alkyl, C ３ -C ７ a compound, wherein the group is selected from cycloalkyl and heterocyclyl; or a stereoisomer or a pharmaceutically acceptable salt thereof.

2. 10. A compound of formula (I) according to claim 1, In the formula, Y is selected from the group consisting of compound.

3. 10. A compound of formula (I) according to claim 1, Represented by formula (Ia): wherein X, R1, R2 and R3 are as defined in claim 1. compound.

4. A compound of formula (I) according to claim 3, and A compound selected from:

5. A conjugate of compound (I) as defined in claim 1, represented by a compound of formula (II): During the ceremony, X is a direct bond or —O—; Y is a linear or branched C substituted with at least two hydroxyl groups. １ -C ６ Alkyl, C ３ -C ７ is a group selected from cycloalkyl and heterocyclyl; R1 is -SO ３ H, -COOH, -CONH ２ and -COO-C １ -C ６ a straight or branched C substituted with a group selected from alkyl １ -C ６ is alkyl; R4 is -SO ３ H, —COOH and —CONH—(S) ｍ -C, which is a straight or branched chain substituted with a group selected from １ -C ６ alkyl, wherein S is a spacer; T is a targeting moiety; m is an integer equal to 0 or 1; and R5 is hydrogen, —SO ３ Straight or branched C substituted by H, —COOH or —CONH—Y １ -C ６ Alkyl and the group CONH—(S) ｍ -T, wherein Y, S, T and m are as defined above; wherein at least one of R4 and R5 is CONH—(S) ｍ - Straight or branched C substituted by T １ -C ６ a conjugate, or a stereoisomer or a pharmaceutically acceptable salt thereof.

6. 6. A compound of formula (II) according to claim 5, In the formula, S is -(CH ２ ) ｐ COO-, -(CH ２ CH ２ O) ｐ CH ２ CH ２ COO- and -(CH ２ CH ２ O) ｐ CH ２ CH ２ NH—, wherein p is an integer comprised between 0 and 20; compound.

7. 7. A compound of formula (II) according to claim 5 or 6, wherein T is a targeting moiety selected from the group consisting of a small molecule, a protein, a peptide, a peptidomimetic, an enzyme substrate, an antibody or fragment thereof, and an aptamer; compound.

8. 8. A compound of formula (II) according to claim 7, wherein T is a moiety that interacts with an integrin receptor. compound.

9. Compounds of formula (II) according to any one of claims 5 to 8, Represented by formula (IIa): wherein R1, R4, R5 and X are as defined in claim 5. compound.

10. 10. A compound of formula (II) according to claim 9, and Selected from: compound.

11. A compound according to any one of claims 1 to 10, Compounds for use as fluorescent probes for biomedical optical imaging applications in mammals.

12. 12. A compound for use according to claim 11, The imaging application is aimed at detecting normal tissue and includes angiography, perfusion imaging, biliary imaging, and neuroimaging; compound.

13. 12. A compound for use according to claim 11, The imaging application is aimed at detecting abnormal tissues, including primary tumor lesions, local or distant metastases, or pre-neoplastic lesions, and is performed under NIR radiation; compound.

14. A pharmaceutical diagnostic composition comprising a compound according to any one of claims 1 to 10 and at least one pharmaceutically acceptable carrier or excipient.

15. A diagnostic kit comprising at least one compound according to any one of claims 1 to 10 together with additional adjuvants thereof for carrying out biomedical optical imaging applications.

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